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A peptide ligase and the ribosome cooperate to synthesize the peptide pheganomycin

Nature Chemical Biology volume 11, pages 71–76 (2015)Cite this article

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Abstract

Peptide antibiotics are typically biosynthesized by one of two distinct machineries in a ribosome-dependent or ribosome-independent manner. Pheganomycin (PGM (1)) and related analogs consist of the nonproteinogenic amino acid (S)-2-(3,5-dihydroxy-4-hydroxymethyl)phenyl-2-guanidinoacetic acid (2) and a proteinogenic core peptide, making their origin uncertain. We report the identification of the biosynthetic gene cluster from Streptomyces cirratus responsible for PGM production. Unexpectedly, the cluster contains a gene encoding multiple precursor peptides along with several genes plausibly encoding enzymes for the synthesis of amino acid 2. We identified PGM1, which has an ATP-grasp domain, as potentially capable of linking the precursor peptides with 2, and validate this hypothesis using deletion mutants and in vitro reconstitution. We document PGM1's substrate permissivity, which could be rationalized by a large binding pocket as confirmed via structural and mutagenesis experiments. This is to our knowledge the first example of cooperative peptide synthesis achieved by ribosomes and peptide ligases using a peptide nucleophile.

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Figure 1: Chemical structures of pheganomycins and its biosynthetic gene cluster identified in S. cirratus.
Figure 2: Compounds used for the N-terminus substrate and analysis of the reaction product formed from 4 and the peptide.
Figure 3: Peptides used for nucleophile and analysis of the reaction product formed from 4 and apidaecin.
Figure 4: Overall structure and docking models of PGM1.
Figure 5: Hypothetical biosynthetic machinery of PGM.

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References

  1. Humphries, R.M., Pollett, S. & Sakoulas, G. A current perspective on daptomycin for the clinical microbiologist. Clin. Microbiol. Rev. 26, 759–780 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Arnison, P.G. et al. Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat. Prod. Rep. 30, 108–160 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Koglin, A. & Walsh, C.T. Structural insights into nonribosomal peptide enzymatic assembly lines. Nat. Prod. Rep. 26, 987–1000 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Giessen, T.W. & Marahiel, M.A. Ribosome-independent biosynthesis of biologically active peptides: application of synthetic biology to generate structural diversity. FEBS Lett. 586, 2065–2075 (2012).

    Article  CAS  PubMed  Google Scholar 

  5. Maruyama, C. et al. A stand-alone adenylation domain forms amide bonds in streptothricin biosynthesis. Nat. Chem. Biol. 8, 791–797 (2012).

    Article  CAS  PubMed  Google Scholar 

  6. Kadi, N., Oves-Costales, D., Barona-Gomez, F. & Challis, G.L. A new family of ATP-dependent oligomerization-macrocyclization biocatalysts. Nat. Chem. Biol. 3, 652–656 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. Suzukake-Tsuchiya, K., Hori, M., Shimada, N. & Hamada, M. Mode of action of deoxypheganomycin D on Mycobacterium smegmatis ATCC 607. J. Antibiot. (Tokyo) 41, 675–683 (1988).

    Article  CAS  Google Scholar 

  8. Crone, W.J.K., Leeper, F.J. & Truman, A.W. Identification and characterisation of the gene cluster for the anti-MRSA antibiotic bottromycin: expanding the biosynthetic diversity of ribosomal peptides. Chemical Science 3, 3516–3521 (2012).

    Article  CAS  Google Scholar 

  9. Huo, L., Rachid, S., Stadler, M., Wenzel, S.C. & Muller, R. Synthetic biotechnology to study and engineer ribosomal bottromycin biosynthesis. Chem. Biol. 19, 1278–1287 (2012).

    Article  CAS  PubMed  Google Scholar 

  10. Hubbard, B.K. & Walsh, C.T. Vancomycin assembly: nature's way. Angew. Chem. Int. Edn Engl. 42, 730–765 (2003).

    Article  CAS  Google Scholar 

  11. Donia, M.S., Ravel, J. & Schmidt, E.W. A global assembly line for cyanobactins. Nat. Chem. Biol. 4, 341–343 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Chen, H., Tseng, C.C., Hubbard, B.K. & Walsh, C.T. Glycopeptide antibiotic biosynthesis: enzymatic assembly of the dedicated amino acid monomer (S)-3,5-dihydroxyphenylglycine. Proc. Natl. Acad. Sci. USA 98, 14901–14906 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Anzai, H. et al. Replacement of Streptomyces hygroscopicus genomic segments with in vitro altered DNA sequences. J. Antibiot. (Tokyo) 41, 226–233 (1988).

    Article  CAS  Google Scholar 

  14. Fawaz, M.V., Topper, M.E. & Firestine, S.M. The ATP-grasp enzymes. Bioorg. Chem. 39, 185–191 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Yamaguchi, H. et al. Three-dimensional structure of the glutathione synthetase from Escherichia coli B at 2.0 Å resolution. J. Mol. Biol. 229, 1083–1100 (1993).

    Article  CAS  PubMed  Google Scholar 

  16. Fan, C., Moews, P.C., Shi, Y., Walsh, C.T. & Knox, J.R. A common fold for peptide synthetases cleaving ATP to ADP: glutathione synthetase and D-alanine:D-alanine ligase of Escherichia coli. Proc. Natl. Acad. Sci. USA 92, 1172–1176 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Tabata, K., Ikeda, H. & Hashimoto, S. ywfE in Bacillus subtilis codes for a novel enzyme, L-amino acid ligase. J. Bacteriol. 187, 5195–5202 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kino, K., Arai, T. & Tateiwa, D. A novel L-amino acid ligase from Bacillus subtilis NBRC3134 catalyzed oligopeptide synthesis. Biosci. Biotechnol. Biochem. 74, 129–134 (2010).

    Article  CAS  PubMed  Google Scholar 

  19. Taguchi, S., Mita, K., Ichinohe, K. & Hashimoto, S. Targeted engineering of the antibacterial peptide apidaecin, based on an in vivo monitoring assay system. Appl. Environ. Microbiol. 75, 1460–1464 (2009).

    Article  CAS  PubMed  Google Scholar 

  20. Holm, L. & Sander, C. Dali: a network tool for protein structure comparison. Trends Biochem. Sci. 20, 478–480 (1995).

    Article  CAS  PubMed  Google Scholar 

  21. Sampei, G. et al. Crystal structure of glycinamide ribonucleotide synthetase, PurD, from thermophilic eubacteria. J. Biochem. 148, 429–438 (2010).

    Article  CAS  PubMed  Google Scholar 

  22. Thoden, J.B., Holden, H.M., Paritala, H. & Firestine, S.M. Structural and functional studies of Aspergillus clavatus N5-carboxyaminoimidazole ribonucleotide synthetase. Biochemistry 49, 752–760 (2010).

    Article  CAS  PubMed  Google Scholar 

  23. Thoden, J.B., Firestine, S.M., Benkovic, S.J. & Holden, H.M. PurT-encoded glycinamide ribonucleotide transformylase. J. Biol. Chem. 277, 23898–23908 (2002).

    Article  CAS  PubMed  Google Scholar 

  24. Suzuki, M. et al. The structure of L-amino-acid ligase from Bacillus licheniformis. Acta Crystallogr. D Biol. Crystallogr. 68, 1535–1540 (2012).

    Article  CAS  PubMed  Google Scholar 

  25. Wang, W., Kappock, T.J., Stubbe, J. & Ealick, S.E. X-ray crystal structure of glycinamide ribonucleotide synthetase from Escherichia coli. Biochemistry 37, 15647–15662 (1998).

    Article  CAS  PubMed  Google Scholar 

  26. Bateman, A. et al. The Pfam protein families database. Nucleic Acids Res. 28, 263–266 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Teixeira, L.K. & Reed, S.I. Ubiquitin ligases and cell cycle control. Annu. Rev. Biochem. 82, 387–414 (2013).

    Article  CAS  PubMed  Google Scholar 

  28. Mazmanian, S.K., Liu, G., Ton-That, H. & Schneewind, O. Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science 285, 760–763 (1999).

    Article  CAS  PubMed  Google Scholar 

  29. Kane, P.M. et al. Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H+-adenosine triphosphatase. Science 250, 651–657 (1990).

    Article  CAS  PubMed  Google Scholar 

  30. Chan, A.O. et al. Modification of N-terminal α-amino groups of peptides and proteins using ketenes. J. Am. Chem. Soc. 134, 2589–2598 (2012).

    Article  CAS  PubMed  Google Scholar 

  31. Ong, S.E. & Mann, M. Mass spectrometry-based proteomics turns quantitative. Nat. Chem. Biol. 1, 252–262 (2005).

    Article  CAS  PubMed  Google Scholar 

  32. Wagner, A.M. et al. N-terminal protein modification using simple aminoacyl transferase substrates. J. Am. Chem. Soc. 133, 15139–15147 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Pan, Y. et al. N-terminal labeling of peptides by trypsin-catalyzed ligation for quantitative proteomics. Angew. Chem. Int. Edn Engl. 52, 9205–9209 (2013).

    Article  CAS  Google Scholar 

  34. Jursic, B.S., Neumann, D. & McPherson, A. Preparation of N-formamidinylamino acids from amino and formamidinesulfinic acids. Synthesis 32, 1656–1658 (2000).

    Article  Google Scholar 

  35. Zerbino, D.R. & Birney, E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18, 821–829 (2000).

    Article  Google Scholar 

  36. Noguchi, H., Taniguchi, T. & Itoh, T. MetaGeneAnnotator: detecting species-specific patterns of ribosomal binding site for precise gene prediction in anonymous prokaryotic and phage genomes. DNA Res. 15, 387–396 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Vara, J., Lewandowska-Skarbek, M., Wang, Y.G., Donadio, S. & Hutchinson, C.R. Cloning of genes governing the deoxysugar portion of the erythromycin biosynthesis pathway in Saccharopolyspora erythraea (Streptomyces erythreus). J. Bacteriol. 171, 5872–5881 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Hopwood, D.A. et al. Gene manipulation of Streptomyces: a Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, 1985).

  39. Rice, L.M., Earnest, T.N. & Brunger, A.T. Single-wavelength anomalous diffraction phasing revisited. Acta Crystallogr. D Biol. Crystallogr. 56, 1413–1420 (2000).

    Article  CAS  PubMed  Google Scholar 

  40. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Afonine, P.V. et al. Towars automated crystallographic structure refinement with phenix. refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  44. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  PubMed  Google Scholar 

  45. Brunger, A.T. Version 1.2 of the crystallography and NMR system. Nat. Protoc. 2, 2728–2733 (2007).

    CAS  PubMed  Google Scholar 

  46. Trott, O. & Olson, A.J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. J. Comput. Chem. 31, 455–461 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This study was supported by a Grant-in-Aid for Scientific Research (23108101 and 25560397 to T.D. and 25108710 to H.M.) from the Japan Society for the Promotion of Science. We thank M. Igarashi from the Institute of Microbial Chemistry and S. Taguchi from Hokkaido University for providing us with S. cirratus and apidaecin, respectively.

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Authors and Affiliations

  1. Graduate School of Engineering, Hokkaido University, Hokkaido, Japan

    Motoyoshi Noike, Koichi Ooya, Ikuo Sasaki, Yasuharu Satoh, Hajime Ito & Tohru Dairi

  2. Institute of Natural Medicine, University of Toyama, Toyama, Japan

    Takashi Matsui, Shouta Ohtaki & Hiroyuki Morita

  3. Department of Bioscience, Fukui Prefectural University, Fukui, Japan

    Yoshimitsu Hamano & Chitose Maruyama

  4. National Institute of Infectious Diseases, Tokyo, Japan

    Jun Ishikawa

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Contributions

M.N., H.M. and T.D. designed the research. M.N., T.M. and K.O. performed in vitro experiments. T.M., S.O. and H.M. carried out crystallography. I.S. and H.I. synthesized substrates and performed NMR studies. M.N., Y.H., C.M. and Y.S. analyzed products by LC/MS. J.I. and T.D. identified the gene cluster. M.N., H.M. and T.D. analyzed data and wrote the manuscript.

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Correspondence to Hiroyuki Morita or Tohru Dairi.

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Supplementary Results, Supplementary Tables 1–8 and Supplementary Figures 1–39. (PDF 4443 kb)

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Noike, M., Matsui, T., Ooya, K. et al. A peptide ligase and the ribosome cooperate to synthesize the peptide pheganomycin. Nat Chem Biol 11, 71–76 (2015). https://doi.org/10.1038/nchembio.1697

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